IE50856B1 - Method of producing n-benzyloxycarbonyl-l-aspartic acid - Google Patents

Abstract

The reaction between L-aspartic acid and benzyloxycarbonyl chloride is carried out at a specific pH range, i.e., 12.0 to 13.5, whereby highly pure N-benzyloxycarbonyl-L-aspartic acid can be obtained in high yields, while both the by-production of N-benzyloxycarbonyl- alpha - or beta -L-aspartyl-L-aspartic acid and the decomposition of benzyloxycarbonyl chloride to benzyl alcohol are suppressed.

Description

This invention relates-to a method of producing N-benzyloxycarbonyl-L-aspartic acid (or a salt thereof).

Various abbreviations are used herein, these abbreviations having the following meanings: Z-Asp means N-benzyloxycarbonyl-L-aspartie acid; a-APA means o-L-aspartyl-L-phenylalanine lower alkyl ester; Asp means L-aspartic acid; Z-AA means N-benzyloxycarbonyl-a-(and/or S-)Laspartyl-L-aspartic acid; Z-AA(a)n means N-benzylOxycsrbonyl-o-L-aspartylL-aspartic acid; Ζ-ΑΑ(β) means N-benzyloxycarbonyl-S-L-aspartylL-aspartic acid; means benzyloxycarbonyl; AAPA means L-aspartyl-L-aspartyl-L-phenylalanine lower alkyl ester; “o-APM means o-L-aspartyl-L-phenylalanine methyl ester; Z-Cl means benzyloxycarbonyl chloride; PA means L-phenylalanine lower alkyl ester; EDC means ethylene dichloride.

The term lower alkyl means an alkyl group containing not more than 4 carbon atoms.

It is well known that g-APAs are useful as sweetening agents. There have been developed various methods of producing a-AFA. One of these methods, disclosed in U.S. Fatent Specification No. 3,786,039, comprises subjecting Asp to benzyloxycarbonylation, and reacting the Z-Asp, after converting it to the corresponding anhydride, with PA. This method is excellent in its ease of operation and its stable reaction, but is defective in that 2-AA is produced as a by-product during the benzyloxycarbonylation.

The Z-AA reacts with PA in the subsequent step, giving an M-benzylOxycsrbonyl-L-aspartyl-L-sspartyl-L50856 phenylalanine lower alkyl ester. Removal of the Z group from the last-mentioned ester gives AAPA, which is very difficult to remove from the a-APA.

In other words, a-APA contaminated by AAPA is very difficult to purify. An ion-exchanger resin has heretofore been necessary to remove the AAPA impurity from a-APA contaminated with AAPA. It is to be noted that a-APM has been already put on the market as a sweetening agent in some countries, namely France, Belgium and Luxembourg.

According to the present invention, there is provided a method of producing Z-Asp Lor a salt thereof), which Comprises reacting Asp for a salt thereof J with Z-Cl while the pH is maintained within the range of from 12.0 to 13.5.

The present invention also provides a-APA produced by reacting Z-Asp (or a salt thereof) produced by the method of the invention, after conversion to the anhydride thereof, with PA.

The inventors of the present invention have found as a result of intensive and extensive study that, if the pH at which Asp is reacted With 2-Cl Is adjusted within a range of 12.0 to 13.5 throughout the reaction time,-both the by-production of Z-AA(,«J and/or Z-AA(£) and the decomposition of Z-Cl to benzyl alcohol are suppressed. Such suppression, in turn, means that highly pure Z-Αερ can be obtained in high yields.

It will be appreciated from the foregoing that, when a-APA is produced by the method described in U.S. Patent Specification No. 3,786,039, highly pure α-APA can be obtained in high yields if Z-Asp obtained in accordance with this invention is used as one of the starting materials.

Ihere are disclosed, for example in Berichte 65, 1192 (1932), methods of producing N-benzyloxycarbonyl-amino acids;including Z-Asp, which comprise reacting various amino acids, including Asp, using 2-Cl. According to the Berichte disclosure, sodium hydroxide or magnesium oxide is used ia Qn amount equivalent or nearly equivalent to the total of (a) carboxyl groups in the free form of the amino acid aDd (b) hydrogen chloride formed from the 2-C1 when the reaction is carried out. Khen Z-Asp is produced under the Serichte reaction conditions, the pH never reaches as high a value as 12.0 (which is the minimum pH used according to this invention) if magnesium oxide is used, and accordingly Z-AA is by-produced in large amounts On the other hand, if sodium hydroxide is used, it is thought that the pH is sometimes elevated locally in the reaction mass to value above 12.0, because there is no suggestion in the Berichte disclosure of maintaining the pH within a certain range throughout the reaction time.

Too high pH values caused locally in the reaction mass bring about the useless decomposition of Z-Cl to benzyl alcohol, and accordingly large amounts of Asp remain unreacted. Even when sodium hydroxide is used, Z-AA is produced in larger amounts than when magnesium oxide is used, unless the pH is sufficiently high.

D.S. Patent Specification No. 3,808,190 discloses in Example 1(A), the preparation of N-carbobenzoxyaspartic acid, according to which the pH was maintained at 8-9 first with sodium bicarbonate and then with sodium hydroxide. This prior art does not hint at the underlying idea of the use of a pH within a specific range, as in the present invention.

By employing the pH conditions of this invention, both the by-production of Z-AA resulting from too low pH values and the by-production of benzyl alcohol resulting from too high pH values are suppressed. Z-Asp can be produced in high yields accordingly.

According to this invention, Asp may be used in the free form or in the salt form such as sodium or potassium salt. Furthermore, it need not be in excessively purified form. It may contain impurities in small amounts insufficient to inhibit the reaction, i.e. the benzyloxycarbonylation. For example, there may be used crude Asp crystals which have been obtained by adjusting tbe pB of an Asp fermentation broth after concentrating if necessary to tbe isoelectric point of Asp, the broth having been obtained by a fermentation method by the use of sugars, fumaric acid or tbe like as raw materials.

There may be used Asp or its monoalkali or dialkali metal salt crystals or tbe like which have been collected from an eluate after concentrating and/or adjusting its pB if necessary, the eluate having been obtained by passing an Asp fermentation broth through a column of a strongly acidic cation exchange resin and eluting the Asp adsorbed on to tbe column with aqueous NaOH solution or the like.

Asp or its salt is usually subjected to benzyloxycarbonylation as its aqueous solution. The solution is adjusted to a pH of from 12.0 to 13.5, using, for example, sodium or potassium hydroxide. Tbe concentration of Asp in the solution is not critical; 20 to 45 weight % solutions are usually used. Too dilute solutions can cause difficulties such as low separation yields of the required product from the reaction mixture. It is desirable that the reaction be carried out in a liquid phase, and accordingly the upper limit of the concentration of Asp in solution is determined by its solubility. 2-C1 may be used as such, but it is usually used as a solution in an organic solvent which does not interfere with tbe reaction and is not miscible with aqueous Asp solution. Examples of the solvent are toluene and EDC. It is appropriate to use Z-Cl in a molar amount of about 1.1 times the molar amount of Asp (having regard to the decomposition of Z-Cl during the reaction period of time) and in a concentration of 30 to 60 weight %. Too low concentrations cause delayed reaction rates, and, on the other hand, too high concentrations prevent efficient removal with an organic solvent of by-products, such as benzyl alcohol, formed in the reaction mixture.

The reaction is preferably carried out by mixing an Asp solution and a Z-Cl solution. Reaction when carried out at elevated temperatures facilitates the decomposition of Z-Cl, and it is appropriate that the reaction be carried out at 0 to 30°C. Stirring may be carried out during the g reaction period, whereby the two solutions are well mixed. The pH of the reaction mass is maintained within a range of 12,0 to 13.5 throughout the reaction period by addition . of, for example, sodium hydroxide. A reaction time of 2 to 3 hours will usually suffice.

When the reaction has finished, Z-Asp, Z-AA, unreacted Asp and the like are present in the aqueous layer, while unreacted Z-Cl, benzyl alcohol resulting from the decomposition of Z-Cl, and the like are present in the organic solvent layer.

The Z-Asp is used as such or after being separated and purified, depending upon its use. One example of a suitable separation and purification method comprises separating the aqueous layer from the organic solvent layer, neutralizing the separated aqueous layer with, for example, hydrochloric acid or sulphuric acid, cooling the neutralized aqueous layer whereby Z-Asp crystallizes out, and separating the Z-Asp crystals. Recrystallization may be carried out, if necessary. From the separated aqueous layer, the Z-Asp may also be separated by extracting with, for example, ethyl acetate, n-butanol and ketones immiscible with water (such as methyl ethyl ketone and methyl isobutyl ketone). Combined recrystallization and extraction technique bring about higher purities.

In accordance with the method of this invention, the by-production of Z-AA, an obstacle to the production of α-APAs, may be suppressed and the racemization of Asp does not take place even at elevated temperature.

Accordingly, highly pure Z-Asp is produced in high yields. This means that the final substance, i.e. an α-APA, can be produced in high yields.

The invention will how be illustrated by the following Example. 8 5« EXAMPLE L-aspartic acid was dissolved in aqueous NaOH solution to produce a 36 weight % Asp solution, and *222g ofthe solution (containing 0.6M Asp) were put into a four-necked flask of one litre capacity, equipped with a stirrer, a thermometer, a pH-meter and a dropping funnel.

The solution was cooled to 10°C, and then 267 g of a 42 weight % solution of Z-Cl in toluene (containing 0.66M Z-Cl) were added. Reaction was carried out at 10-30°C for 3 hours, while the pH of the reaction mixture was maintained within a particular range by the addition of aqueous 25% NaOH solution.

The toluene was thereafter removed by layer separation. To the aqueous layer, 230 g of water were added. The pH was adjusted to 1 with 35% hydrochloric acid, and the solution was allowed to stand overnight at 5°C.

The resulting Z-Asp crystals were collected by centrifugation, washed with water and dried under reduced pressure.

Six runs of the above procedure were carried out at various pH ranges. The results are listed in the following Table. Analysis of the impurities was carried out hy thin layer chromatography (100 γ spot).

TABLE Run No. 1 2 3 4 5 6 25 j® range (not less than ~ less than) 8.0 ~10.0 10.0 «*11.0 11.0 ~ 12.0 12.0 ~13.0 12.5 -13.5 13.5 —' 30 Yield in mole % of 2-Asp based on Asp 68.3 79.5 78.6 87.3 86.2 80.2 Inpurities in Z-Asp crystals Z-AA(a) Ζ-ΑΑ(β) 3.5 1.5 3.0 1.0 1.5 0.5 Trace Trace Trace TYace Not detected Not detected 35 (%) Asp 5.0 0.2 0.3 0.6 1.0 7.5 others not detec- ted not detec- ted not detec- ted _ not detec- ted not detec- ted Trace 7 It can be seen from the Table that highly pure Z-Asp can be produced in high yields under the reaction conditions of this invention.

Claims (6)

1. A method of producing N-benzyloxycarbonyl-Laspartic acid (or a salt thereof), which comprises reacting L-aspartic acid (or a salt thereof) with benzyloxycarbonyl chloride while the pH is maintained witnin the range of from 12.0 to 13.S.

2. A method according to claim 1, wherein the L-aspartic acid (or salt thereof) is used in the form of an aqueous solution thereof, and wherein the benzyloxycarbonyl chloride is used as such or in the form of a solution thereof in an organic solvent which does not interfere with the reaction and which is not miscible with the aqueous solution of L-aspartic acid (or salt thereof).

3. A method according to claim 1 or 2, wherein the rection is carried out at a temperature of from 0 to 30’C.

4. A method according to claim 1, substantially as described in the foregoing Example.

5. N-benzyloxycarbonyl-L-aspartic acid (or a salt thereof), produced by a method according tc any of claims 1 to 4.

6. An a-L-aspartyl-L-phenylalanine lower alkyl ester, produced by reacting N-benzyloxyCarbonyl-Laspartic acid (or a salt thereof) as claimed in claim 5, after conversion to the anhydride thereof, with an L-phenylalanine lower alkyl ester.